Clones of serogroup B Neisseria meningitidis causing systemic disease in The Netherlands, 1958-1986.

Serogroup B isolates of Neisseria meningitidis recovered from 278 patients with systemic disease in the Netherlands between 1958 and 1986 were analyzed with respect to serotype and multilocus enzyme genotype. Of the isolates, 28% were serotype 2b and 53% were neither serotypeable nor serosubtypeable. There were 145 distinct multilocus genotypes (electrophoretic types, ETs), with up to 31 isolates belonging to the same ET. Temporal changes in the genotypic composition of meningococcal populations in the Netherlands were demonstrated by the recent occurrence of disease caused by three clone lineages, I, III, and VI, that were not found before 1975. The epidemic of 1966-1967 and the hyperendemic wave of 1972 were caused, in large part, by two closely related but distinct clones of serotype 2b isolates, ET-11 and ET-17, respectively. Deviations in male-to-female ratio and age distribution of patients were observed for disease caused by isolates of individual clone lineages.

The epidemiology of Haemophilus influenzae meningitis in children under five years of age in The Gambia, West Africa.

Haemophilus influenzae meningitis was studied over a 2-year period in children less than 5 years of age in a population-based, prospective study in The Gambia, West Africa. A total of 77 cases were recorded. The incidence was 60 cases/100,000 children less than 5 years of age and 297 cases/100,000 children less than 1 year of age. The peak prevalence was at the age of 5 months; 83% of the children were less than 1 year old and 45% were less than 6 months old. Only 55% of cases recovered completely. The distance to the nearest hospital was an important predisposing factor for a fatal outcome. Two cases were secondary. Many patients were anemic and underweight. The observations show that H. influenzae meningitis in The Gambia has an incidence as high as that in the USA, but that it has 10-fold more devastating outcome. If the infection is to be prevented by vaccination in The Gambia, immunization will have to be given very early in life.

Haemophilus influenzae infections in patients with chronic obstructive pulmonary disease despite specific antibodies in serum and sputum.

The titer and specificity of antibodies to the infecting Haemophilus influenzae was determined in sera and sputa from 27 patients with chronic obstructive pulmonary disease (COPD) to analyze the specific immune response. COPD patients had significantly higher serum IgG and IgA antibody titers than 13 healthy controls (mean IgG titers 12,302 and 5,623, respectively; mean IgA titers, 2,398 and 912; p less than 0.001). The mean IgM titers were comparable: 501 and 447, respectively. Specific IgA antibodies were also detectable in the sputum of the COPD patients (mean IgA antibody titer, 776). The local antibody production was determined by calculating the relative coefficient of excretion (RCE) to albumin. The mean RCE of 89.1 for IgA indicated statistically significant local production (p less than 0.02), in contrast to a nonsignificant increase for IgG (mean RCE of 3.6). Specific IgM was below the detection level. Immunoblotting experiments showed that the antibodies in sera from COPD patients and controls were directed against most of the outer membrane proteins of H. influenzae, with individual differences between IgG, IgA, and IgM. The IgA and IgG antibodies in serum had a similar specificity as those in sputum. The appearance or persistence of H. influenzae coincided with minor changes in antibody titer and specificity. From these results we conclude that COPD patients are infected with H. influenzae despite the presence of at least as many antibodies in sputum and serum as in controls and that these antibodies are directed against a variety of antigenic determinants of the infecting strain.

Endogenous and exogenous reinfections by Haemophilus influenzae in patients with chronic obstructive pulmonary disease: the effect of antibiotic treatment on persistence.

To analyze whether exacerbations in chronic obstructive pulmonary disease (COPD) coincide with reinfection by Haemophilus influenzae, 16 COPD patients were studied longitudinally for 3 years. Exacerbations coincided with reinfection by H. influenzae, either endogenous, by a strain with a DNA fingerprint indistinguishable from the strain previously present but with another major outer membrane protein (MOMP) pattern (2 patients), or exogenous, by a strain with a different DNA fingerprint and MOMP pattern (3 patients). The other patients, remaining in an infectious state without clear exacerbations for longer periods, were persistently infected by a particular H. influenzae strain (median persistence time, 5.5 months; range, 2-23 months). Of 8 antibiotic-treated patients, 7 remained infected by H. influenzae with the same DNA fingerprint, although all strains were sensitive to the antibiotics prescribed. Results of the study suggested that exacerbations in COPD patients coincide with endogenous or exogenous reinfection by H. influenzae, persistently infected patients keep the same H. influenzae strain for longer periods, and antibiotic treatment was not effective in eradicating H. influenzae.

Carriage of Haemophilus influenzae in healthy Gambian children.

1240 throat samples were processed during different seasons in 11 different communities of The Gambia (West Africa). The carriage rate for Haemophilus influenzae type b ranged from 0 to 33%, but often attained 10% or more, higher than that reported from other open communities. The duration of carriage was short (less than 3 months) and H. influenzae b was found in only 10% of the carriers isolated during the previous or the following survey. Children less than 5 years old carried H. influenzae b in their throat significantly more often than children older than 14 years (P less than 0.05). A high carriage rate did not correlate with the wet or dry season. The carriage rate of children in rural areas was similar to that of children in urban areas. Children in day-care centres or nurseries had a surprisingly low carriage rate (2%). The carriage rate of H. influenzae b was compared to the presence of H. influenzae subspecies in a random sample, which revealed that H. influenzae subspecies was found in 90% of the children under 5 years old. Encapsulated strains of H. influenzae were found in 25% of the same sample, two-thirds of which were not type b. All capsule types were represented. No meningitis cases occurred in the survey populations. We conclude that the prevalence of H. influenzae b in open Gambian communities is similar to that in closed communities elsewhere, but that the kinetics are different from those in closed communities, as persistence of infection in Gambian children is short-lived.

Nonculture detection of Haemophilus influenzae in sputum with monoclonal antibodies specific for outer membrane lipoprotein P6.

Isolation of Haemophilus influenzae from sputum is hampered by overgrowth by other microorganisms or by antibiotic treatment of the patient. To overcome this problem in the detection of H. influenzae, an in situ immunoperoxidase staining technique was developed with monoclonal antibody (MAb) 8BD9, immunoglobulin subclass G2a. MAb 8BD9 appeared to be directed to an epitope on the outer membrane lipoprotein P6 of H. influenzae. The species specificity of MAb 8BD9 was analyzed by staining isolates from different bacterial species. MAb 8BD9 reacted with all 300 H. influenzae strains tested and with H. aegyptius and H. haemolyticus. Twenty-six of 30 H. parainfluenzae strains, other Haemophilus species, and other bacterial species often isolated from sputum were not stained. The staining technique was compared with culture of 845 routinely obtained sputum samples from patients with respiratory tract infections. In 829 sputa (98.1%), the results of both techniques were in agreement; 173 were positive for H. influenzae, and 656 were negative. With 14 sputum samples, the staining method gave a positive result for H. influenzae, but the bacterium was not cultured. This could be ascribed to antibiotic treatment of the patient (n = 7), the presence of other MAb 8BD9-positive Haemophilus species in the sputum (n = 5), and overgrowth by swarming Proteus mirabilis or by Branhamella catarrhalis. In the immunoperoxidase- and Gram-stained smears of two sputum samples, no bacteria were seen, although some H. influenzae was cultured. On the basis of these results, we conclude that immunoperoxidase staining with MAb 8BD9 is a fast and reliable extension of the available detection techniques for H. influenzae.

Antigenic drift of Haemophilus influenzae in patients with chronic obstructive pulmonary disease.

Differences in the major outer membrane protein b,c (molecular weight, 39,000 to 41,000) of related Haemophilus influenzae strains isolated from the sputum of patients with chronic obstructive pulmonary disease were analyzed biochemically and immunologically. Protein b,c was isolated from a total of six related H. influenzae strains from two chronic obstructive pulmonary disease patients. After CNBr digestion of the proteins, the differences in size appeared in the larger of the two fragments. Trypsin and chymotrypsin digests of proteins from related H. influenzae strains showed that proteins differed by only a few peptides or were very similar, in contrast to the peptide maps of proteins from nonrelated strains. Peptide analysis of b,c proteins from related H. influenzae strains by high-performance liquid chromatography after Staphylococcus aureus V8 protease digestion and amino acid analysis of corresponding fractions revealed highly comparable patterns, indicating only minor differences in the amino acid sequences of these proteins. Immunization of rabbits with intact bacteria of four related H. influenzae strains resulted in a strong anti-protein b,c antibody response in all animals. In three of four rabbits, antibodies specific for the b,c protein of the strain used for immunization were elicited, indicating that the changed proteins contained specific immunodominant epitopes. Anti-protein b,c antibodies promoted strain-specific, complement-dependent, bactericidal activity. From these results, we conclude that H. influenzae shows antigenic drift in immunodominant epitopes, caused by small changes in amino acid composition of the b,c protein. Antibodies to these epitopes promote complement-dependent bactericidal activity.

Clinical and epidemiologic aspects of members of Aeromonas DNA hybridization groups isolated from human feces.

Between June 1982 and May 1987 Aeromonas species were isolated from 208 of 34,311 (0.61%) fecal samples submitted to a Regional Public Health Laboratory in The Netherlands. Aeromonas isolates were found most frequently in summer and rarely in winter. Of 169 Aermonas isolates that were available for further study, 19% were isolated from patients with a mixed infection, 5% from patients with underlying diseases, and 15% from patients who used medication that could predispose the intestinal tract to colonization with Aeromonas species. Aeromonas species that produced cytotoxins to Vero cells (cytotoxigenic) were found in hybridization groups 1 (11% of all isolates), 2 (1%), 3 (2%), and 8 (25%) and were identified phenotypically as A. hydrophila or A. sobria. Aeromonas species that did not produce cytotoxins to Vero cells (noncytotoxigenic) were found in hybridization groups 4 (57%) and 5A (4%) and were identified phenotypically as A. caviae. Distribution of Aeromonas species by age showed a predominance of noncytotoxigenic strains in children under the age of 5 years (46% of all noncytotoxigenic strains), while cytotoxigenic strains were mainly cultured from patients aged 50 years or older (54% of all cytotoxigenic strains). Significant correlations were found between cytotoxigenic strains and hospitalization, foreign travel, and contact with surface water. Cytotoxigenic strains were isolated significantly more often than noncytotoxigenic strains from patients with diarrhea, but in a multivariate analysis including age, previous medication, underlying disease, and foreign travel, this association was not significant.

Typing of Aeromonas strains by DNA restriction endonuclease analysis and polyacrylamide gel electrophoresis of cell envelopes.

The outer membrane protein (OMP) composition (OMP typing) of 46 fecal Aeromonas strains from hybridization groups (HGs) 1 (A. hydrophila; n = 10), 4 (A. caviae; n = 16), and 8 (A. veronii; n = 20) were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a phenotypic typing method. Almost every isolate of HG-1 and HG-8 had a unique OMP profile, in contrast to isolates of HG-4, which were separated into five different OMP types. It was possible to recognize HGs 1, 4, and 8 by OMP profiles. Twenty-three Aeromonas strains from HGs 1 (n = 5), 4 (n = 10), and 8 (n = 8) were tested by whole-cell DNA restriction endonuclease analysis (REA) as a genetic typing method. All strains tested by REA (with SmaI) had different DNA digestion patterns. Although additional DNA-rRNA hybridization analyses with SmaI and 16S and 23S rRNAs from Escherichia coli showed a reduction in the number of restriction bands to 8 to 13 hybridized fragments, the discriminative value was less when compared with that obtained by REA. The individual differences found by REA were used to analyze whether patients remained colonized by the same Aeromonas strain. Of 11 patients with diarrhea, 2 had a different isolate on repeat culture. In addition, one of nine tested fecal samples contained two Aeromonas isolates with different REA patterns. These results indicate that during diarrheal disease the intestinal tract may be colonized simultaneously with different Aeromonas isolates.

Antimicrobial susceptibility of sixty human fecal isolates of Aeromonas species.

The MICs of 21 antimicrobial agents were determined for 60 strains of Aeromonas spp. isolated from human feces. All isolates tested were susceptible to aztreonam, tetracycline, imipenem, moxalactam, pipemidic acid, gentamicin, trimethoprim-sulfamethoxazole, pefloxacin and ciprofloxacin. Resistance to erythromycin and streptomycin was observed in all 60 strains. Aeromonas caviae was less susceptible to cefamandole, cefotaxime, norfloxacin, chloramphenicol, tetracycline, sulfamethoxazole and trimethoprim than was either Aeromonas hydrophila or Aeromonas sobria. It was concluded that cotrimoxazole or one of the newer quinolones can be considered for treatment of aeromonas-associated diarrhea.

Rapidly decreased serum IgG to Campylobacter pylori following elimination of Campylobacter in histological chronic biopsy Campylobacter-positive gastritis.

The anaerobic bacterium Campylobacter pylori (Cp) is thought to be associated with chronic gastritis. This paper presents clinical data underpinning this view. Five patients with histological chronic gastritis as determined by diagnostic endoscopy, which was associated with Cp as determined by positive biopsy cultures, all possessed statistically raised serum IgG ELISA titers to Cp during a longitudinal period of observation of 15 months. Treatment with the antibiotics amoxycillin (clamoxyl) or colloidal bismuth subcitrate (denol) eliminated Cp within one month. Associated with this, serum IgG ELISA titers were found to decrease sharply and rapidly. Tagamet and spiramycin had little effect. Although the data are preliminary, they support the assumed Cp involvement in chronic gastritis and suggest that specific serum IgG ELISA titers to Cp are useful parameters in monitoring disease status, exceeding bacteriological culture of biopsy specimens in speed and convenience.

Phenotypic characterization and DNA relatedness in human fecal isolates of Aeromonas spp.

Phenotypic characteristics were used to identify 189 Aeromonas strains isolated from human feces. One hundred forty-two of these strains were placed in 11 DNA hybridization groups, and the genetic and phenotypic data were compared. According to the criteria of Popoff, 66% of the strains were identified as Aeromonas caviae, 18% were identified as A. sobria, and 16% were identified as A. hydrophila. Some biochemical characteristics differed from the criteria of Popoff; 19 of 40 (48%) of tested strains were encapsulated, 42 of 124 (34%) of A. caviae strains were nonmotile, and all A. sobria strains were resistant to KCN. Gas production from D-glucose was temperature dependent; 11 of 64 (17%) A. hydrophila and A. sobria strains produced gas only at 22 degrees C. Of 142 Aeromonas strains, 57% belonged to hybridization group 4, 25% belonged to group 8, 11% belonged to group 1, 4% belonged to group 5A, 2% belonged to group 3, and 1% belonged to group 2. Of 26 strains phenotypically identified as A. hydrophila, 8 (31%) were in hybridization group 8, which contains strains of the new species A. veronii. It therefore appears that our ability to identify Aeromonas strains phenotypically is not sufficiently specific. Either additional definitive biochemical markers must be found or phenotypic identification, at least for some Aeromonas groups, must be regarded as only presumptive.

Differences in lipopolysaccharide profiles of serologically identical Legionella pneumophila serogroup 6 strains.

Over five years 18 strains of Legionella pneumophila serogroup 6 were isolated in Amsterdam from the hot water supply in three hospitals and from one patient. Immunodiffusion and immunoblot procedures showed that these strains were identical. Profiles of isolated lipopolysaccharides from the 18 strains and the reference serogroup 6 strain were visualised in polyacrylamide gels stained with silver. Four strains from hospital A, isolated in 1982, 1984, and 1985 displayed similar lipopolysaccharide profiles which were different in relative mobility from those of hospitals B and C. Those from hospital B (12 strains isolated in 1983 and 1986) and C (one strain) were similar in relative mobility but different in colour. The strain from a patient with acquired immune deficiency syndrome (AIDS) in hospital A displayed a lipopolysaccharide profile characteristic of hospital A. These reproducible profiles were all different in relative mobility from the reference serogroup 6 strain. They can be used as a marker system in epidemiological surveys of serologically identical serogroup 6 strains. Lipopolysaccharide patterns from strains isolated throughout the years in the same hospital were similar. This suggests an outgrowth from organisms inhabiting the plumbing system rather than reseeding from the Amsterdam mains supply.

Changes in outer membrane proteins of nontypable Haemophilus influenzae in patients with chronic obstructive pulmonary disease.

Five individual colonies of Haemophilus influenzae were isolated from each of one to three cultures of sputum collected from 18 patients with chronic obstructive pulmonary disease (COPD). The isolates were studied to investigate whether the major outer membrane proteins (MOMPs) changed during persistence. The relationship between isolates was analyzed by fingerprinting their chromosomal DNA. The fingerprints of eight strains (isolated from eight patients) with various MOMP compositions were different, whereas fingerprints of isolates with identical MOMP compositions were indistinguishable. In 12 patients, two or more strains with different MOMP compositions were found; one strain was isolated from the sputum samples of each of the six remaining patients. In seven of the 12 patients, strains with different MOMPs but with indistinguishable fingerprints were found. The differences were found in proteins b,c (five patients) and d (five patients). In patients with COPD, the MOMPs of H. influenzae are subject to changes that may enable this bacterium to escape immunological defense mechanisms.

The interaction between Neisseria gonorrhoeae and the human cornea in organ culture. An electron microscopic study.

Explants of human corneas in organ culture were used to study the interaction between Neisseria gonorrhoeae and human corneal epithelium at an ultrastructural level. scanning electron microscopy revealed that infection of the corneal explants with N. gonorrhoeae resulted in a rapid adherence of the bacteria to the cell surface. This attachment was probably mediated by pili since only piliated strains were able to adhere to the cells. Upon attachment the bacteria appeared to become engulfed by the epithelial cells. Transmission electron microscopy revealed gonococci apparently lying within vacuoles inside the cells within 1 h after inoculation of the bacteria. At prolonged infection (8-24 h), the thickness of the epithelium was found to be considerably reduced. This thinning of the cornea was probably caused by a continuous desquamation of infected cells. Taken together, the present data demonstrate that Neisseria gonorrhoeae is able to adhere and penetrate into intact corneal epithelium and furthermore indicate that human cornea explants in organ culture are a useful model in studies of bacterial-epithelial cell interaction.

Campylobacter pyloridis-associated chronic active antral gastritis. A prospective study of its prevalence and the effects of antibacterial and antiulcer treatment.

To determine the clinical importance of Campylobacter pyloridis infection, its association with gastric inflammation, and the response to drug therapy, patients with a duodenal or gastric ulcer (n = 63), patients with nonulcer dyspepsia (n = 240), and asymptomatic volunteers (n = 34) were studied. In a prospective longitudinal study, the type, intensity, and distribution of inflammation in antral biopsy specimens were correlated with the presence of C. pyloridis. Campylobacter pyloridis was cultured from antral biopsy specimens in 98% of the ulcer patients, 70% of the nonulcer dyspepsia patients, and 20% of the asymptomatic volunteers. The dependency of chronic active gastritis on the presence of C. pyloridis was shown by an association of gastritis with positive culture and healing of gastritis with negative culture after various therapeutic regimens. Spontaneous disappearance of C. pyloridis never occurred. Colloidal bismuth subcitrate, amoxicillin, and the combination of colloidal bismuth subcitrate and amoxicillin were effective therapies in eradicating C. pyloridis. Recolonization with the same bacterial subtype and recurrence of gastritis frequently occurred within 1 mo after initial eradication. In this study we demonstrate ultimate normalization of gastric mucosa after successful eradication of C. pyloridis. Especially complete normalization of gastric mucosa after amoxicillin monotherapy provides additional strong evidence for a true cause-effect relationship between C. pyloridis colonization and gastritis.